Cancer stem cells (CSCs) are considered to be the origins of cancer. The reason is that these cells have the ability to self-renew and differentiate to form a tumor hierarchy (Non-patent Document 1). Furthermore, CSCs can migrate and be tolerant to anti-cancer drug therapy (Non-patent Document 1). Since CSCs are believed to be a rare subset in tumors, they have been characterized based on cell surface markers and tumor-initiating activity in xenograft transplantations. CSCs have been identified and characterized in several types of cancer, including acute myelocytic leukemia (AML) (Non-patent Documents 2 and 3), breast cancer (Non-patent Document 4), glioma (Non-patent Document 5), head and neck cancer (Non-patent Document 6), pancreatic cancer (Non-patent Documents 7 and 8), lung cancer (Non-patent Document 9), prostatic cancer (Non-patent Documents 10 and 11), mesenchymal neoplasm (Non-patent Document 12), and melanoma (Non-patent Documents 13 and 14). Earlier studies of O'Brien et al. (Non-patent Document 15) and Ricci-Vitiani et al. (Non-patent Document 16) showed that CD133 served as a CSC marker for colon cancer. Thereafter, different research groups have reported other markers: CD44, EpCAM, CD 166 (Non-patent Document 17), and ALDH (Non-patent Documents 18 and 19). Recently, Pang et al. demonstrated that CD26 serves as a marker for a CSC subpopulation with metastatic capacity (Non-patent Document 20).
To isolate CSCs, most studies have employed a cell selection approach using in combination CSC markers such as EpCAMhigh/CD44+/CD166+ (Non-patent Document 17), CD133+/CD44+ (Non-patent Document 21), CD44high/ALDH+ (Non-patent Document 18), and ALDH1+/CD133+ (Non-patent Document 19). In vitro spheroid (cell mass) cultures and direct cancer cell xenograft transplantation to immunodeficient mice have also been used to enrich CSCs (Non-patent Document 22). However, the number and purity of stem cells are still insufficient for further understanding the properties of CSCs.
One challenge in isolating CSCs arises from the phenotypic heterogeneity and/or instability of these cells (Non-patent Document 29). Three-dimensional spheroid cultures are often used as a CSC source. Spheroid cultures are applicable directly to primary tumor cells of clinically resected specimens; thus, maintenance of heterogeneous CSC populations can have certain potential advantages compared to xenograft transplantations. Due to the heterogeneity, however, results of biochemical analyses often show complicated CSC characteristics. CSC selection using antibodies against cell surface marker proteins is commonly used to isolate CSCs, but the number and purity of cells obtained by this method is limited. Phenotypes from xenografts remain stable even after frequent passages, and using xenografts as a source of CSCs is also a common approach. However, there is an argument that xenograft passages in mice only select cells viable in mice and result in elimination of cells that are hardly affected by such an environment. It goes without saying that CSCs in xenograft tumors reflect the characteristics of original CSCs, as long as they maintain the self-renewability and lineage differentiation capacity of the original tumor.
Leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) was originally identified as an orphan G-protein-coupled receptor of the glycoprotein hormone receptor family (Non-patent Documents 23 and 24) and was demonstrated to be a Wnt target gene whose expression is restricted to the crypt (Non-patent Document 25). The discovery that Lgr5-positive columnar cells can regenerate all epithelial lineages (Non-patent Document 25) and that a single Lgr5-positive cell can form crypt-villus organoids in vitro without a mesenchymal niche (Non-patent Document 26), conclusively proves that Lgr5-positive cells are stem cells in the normal intestine and colon. It has also been reported that Lgr5-positive cells form adenomas in the absence of Apc (Non-patent Document 27) and Lgr5 is expressed in colon cancer cell lines (Non-patent Document 25). When considered together, the findings described above suggest that Lgr5-positive cells are an origin of intestine and colon cancers (Non-patent Document 25). It has been proven that, as in stem cells of the normal colon and intestine, Wnt activity is essential for in vitro and in vivo proliferation of CSCs and that exogenous HGF enhances Wnt activity (Non-patent Document 28).
Lgr5 was identified as a marker for normal colon and intestine stem cells, and has been demonstrated to serve as a marker for origins of colon cancer (Patent Document 1 and Non-patent Document 30). Furthermore, Lgr5 was reported to be a protein that is over-expressed in colon cancer stem cells (Patent Document 2). The biological role of Lgr5 in the development of colon cancer remains poorly understood.
To date, various anti-cancer drugs and cancer therapeutic methods have been developed, but there are still issues to be solved, such as poor effectiveness, adverse effects, or being effective in only a limited number of patients. In recent years, therapeutic methods for targeting cancer stem cells have drawn attention, but their effectiveness and adverse effects remain poorly understood (Non-patent Document 31).